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目的克隆深圳水库区双脐螺rDNA基因片段并进行序列分析。方法采集深圳水库排洪渠水体的双脐螺,提取基因组DNA,PCR方法扩增r DNA基因片段,纯化后与p MD18-T质粒连接,转化大肠埃希氏菌JM109,筛选阳性克隆;重组质粒经双酶切鉴定后,进行序列测定,并以BLAST和MEGA4软件分析序列特点。结果深圳水库区双脐螺r DNA基因扩增片段大小约为959 bp;重组质粒经双酶切鉴定与预期结果相符;克隆的rDNA序列含有959个碱基,BLAST比对结果显示,rDNA克隆序列与Gen Bank中3株库恩双脐螺(B.kuhniana)序列的同源性为99%,与2株藁杆双脐螺(B.straminea)的同源性为98%,与1株中介双脐螺(B.intermedia)及2株亚马逊双脐螺(B.amazonica)的同源性介于96%~97%。应用邻位连接法(Neighbor-jioning,NJ)和最小进化法(Minimum Evolution,ME)2种方法构建系统发生树,深圳水库区双脐螺与3株B.kuhniana的遗传距离小,同属一个分支。结论成功克隆了深圳水库区双脐螺r DNA基因片段,其基因遗传特征与B.kuhniana双脐螺接近。
Objective To clone and sequence the rDNA gene of Umbilicus in Shenzhen reservoir area. Methods Genomic DNA was extracted from genomic DNA of Shenzhen flood drainage canal. The r DNA gene fragment was amplified by PCR and ligated with pMD18-T plasmid. The recombinant plasmid was transformed into Escherichia coli JM109 and the positive clones were screened. The recombinant plasmid After identification by double enzyme digestion, sequence analysis was performed, and sequence characteristics were analyzed by BLAST and MEGA4 software. Results The amplified fragment of rDNA gene was about 959 bp in Shenzhen reservoir area. The recombinant plasmid was identified by double enzyme digestion and was consistent with the expected results. The cloned rDNA sequence contained 959 bases. BLAST analysis showed that the rDNA clone sequence It shared 99% homology with the three B.kuhniana sequences in Gen Bank and 98% with two strains of B.straminea, The homologies of B.intermedia and B. amazon were between 96% and 97%. Phylogenetic trees were constructed using Neighbor-jioning (NJ) and Minimum Evolution (ME) methods. The genetic distance between the two strains of B. umbellatus and three strains of B.kuhniana in Shenzhen Reservoir was small and both belong to one branch . Conclusion The rDNA gene fragment of B. umbellatus from Shenzhen Reservoir was successfully cloned and its genetic characteristics were close to those of B. kuhniana.